Pharmaceutical compositions of near IR closed chain, sulfo-cyanine dyes

ABSTRACT

Sterile and non-toxic pharmaceutical compositions of near IR, closed chain, sulfo-cyanine dyes and methods for visualizing tissue under illumination with near-infrared radiation are provided.

BACKGROUND OF THE INVENTION

Minimally invasive medical techniques are intended to reduce the amountof extraneous tissue that is damaged during diagnostic or surgicalprocedures, thereby reducing patient recovery time, discomfort, anddeleterious side effects. While millions of “open” or traditionalsurgeries are performed each year in the United States; many of thesesurgeries can potentially be performed in a minimally invasive manner.One effect of minimally invasive surgery, for example, is reducedpost-operative recovery time and related hospital stay. Because theaverage hospital stay for a standard surgery is typically significantlylonger than the average stay for an analogous minimally invasivesurgery, increased use of minimally invasive techniques could savemillions of dollars in hospital costs each year. While many of thesurgeries performed in the United States could potentially be performedin a minimally invasive manner, only a portion currently employ thesetechniques due to instrument limitations, method limitations, and theadditional surgical training involved in mastering the techniques.

Minimally invasive tele-surgical systems are being developed to increasea surgeon's dexterity as well as to allow a surgeon to operate on apatient from a remote location. Telesurgery is a general term forsurgical systems where the surgeon uses some form of remote control,e.g., a servomechanism, or the like, to manipulate surgical instrumentmovements rather than directly holding and moving the instruments byhand. In such a telesurgery system, the surgeon is provided with animage of the surgical site at the remote location. While viewing thesurgical site on a suitable viewer or display, the surgeon performs thesurgical procedures on the patient by manipulating master control inputdevices, which in turn control the motion of instruments. These inputdevices can move the working ends of the surgical instruments withsufficient dexterity to perform quite intricate surgical tasks.

Minimally invasive medical techniques, including tele-surgical systemscan be further aided by improving visualization of the tissue where theprocedure is to be carried out. One way to improve visualization oftissue is through the use of dyes. Thus, there is a need for novel,sterile, and non-toxic pharmaceutical compositions of near IR, closedchain, sulfo-cyanine dyes and methods for the visualization of tissueunder illumination with near-infrared radiation. Surprisingly, thepresent invention meets these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention generally provides novel, sterile, and non-toxicpharmaceutical compositions of near IR, closed chain, sulfo-cyanine dyesand methods for the visualization of tissue under illumination withnear-infrared radiation.

In a first embodiment, the present invention provides a sterilepharmaceutical composition comprising a unit dosage form of a compoundhaving the formula:

wherein,

R¹, R², R³, and R⁴ are each independently hydrogen, C₁₋₃ alkyl, or—CO₂T;

X is a single bond, —O—, or —S—;

subscripts a, b, c, and d are each independently an integer from 1 to 6,and

T is a metal ion, H, or a negative charge;

-   wherein, the composition is sterile, non-toxic, and adapted for    administration to a patient for visualization of tissue under    illumination with near-infrared radiation; and    wherein, the unit dosage form of the compound delivers to the    patient an amount between 0.05 and 8 mg/kg.

In some cases, the composition has the formula:

wherein,

R¹, R², R³, and R⁴ are each independently hydrogen, —CH₃, or —CO₂T.

In some cases, the composition has the formula:

In some cases, the composition unit dosage form delivers to the patientthe amount of 0.05, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55,0.60, 0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8 mg/kg.

In some cases, the composition is adapted for administration to a humanpatient to obtain visualization of human tissue under illumination withnear-infrared radiation wherein the unit dosage form delivers to thepatient an amount between 0.05 and 8 mg/kg. In some cases, thecomposition is dry and a single dose form.

In some cases the composition is lyophilized in a sterile container. Insome cases, the composition is contained within a sterile container,where the container has a machine detectable identifier which isreadable by a medical device.

In some cases, the composition further comprises combination withpharmaceutically acceptable excipients in an oral dosage form. In somecases, the composition further comprises combination withpharmaceutically acceptable carriers in an injectable dosage form. Insome cases, the composition further comprises combination withpharmaceutically acceptable excipients in a dosage form for directapplication to a surgical site.

In some embodiments, the present invention provides a use of thecomposition adapted for administration to a patient to obtainvisualization of tissue under illumination with near-infrared radiationwherein the unit dosage form delivers to the patient an amount between0.05 and 8 mg/kg. In some cases, the use is adapted for administrationto a human patient to obtain visualization of human tissue underillumination with near-infrared radiation wherein the unit dosage formdelivers to the patient an amount between 0.05 and 8 mg/kg.

In some embodiments, the present invention provides a method forvisualization of tissue, the method comprising administering thecomposition to a patient, imaging the tissue under illumination withnear-infrared radiation, and obtaining at least one image of tissue fromthe patient using the composition. In some cases, the method furthercomprises obtaining the image during administration, afteradministration, or both during and after administration of thecomposition.

In some cases, the method further comprises intravenously injecting thecomposition into a patient. In some cases, the composition is injectedinto a circulatory system.

In some cases, the method further comprises visualizing a patient areaon which surgery is or will be performed, or for viewing a patient areaotherwise being examined by a medical professional. In some cases, themethod further comprises performing a surgical procedure on the patientareas based on the visualization of the surgical area. In some cases,the method further comprises viewing a patient area on which anophthalmic, arthroscopic, laparoscopic, cardiothoracic, muscular,reconstructive, or neuro procedure is or will be performed. In somecases, the method further comprises obtaining ex vivo images of at leasta portion of the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the visualization of a ureter after injection of acomposition of formula III on the da Vinci Surgical System uponexcitation with near-infrared light in Firefly fluorescence mode. Uponconversion to greyscale images, quantification of infrared signalintensity of the kidney was measured in white light.

FIGS. 2A and 2B show the ureter of a pig after injection of acomposition of formula III. FIG. 2A shows white light visualization ofthe ureter of a pig after injection of a composition of formula III onthe da Vinci Surgical System. FIG. 2B shows visualization of the ureterof a pig after injection of the composition of formula III on the daVinci Surgical System upon excitation with near-infrared light inFirefly fluorescence mode. Upon conversion to greyscale images,quantification of signal intensity of the ureter (R1, for Region 1) andbackground tissue (R2, for Region 2) were measured in the white light(no near infrared signal, FIG. 2A) and fluorescent (infrared signalpresent, FIG. 2B) images. The signal to background ratio, R1/R2 is 1.22in the white light image and 2.58 in the fluorescent image.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides novel, sterile, and non-toxicpharmaceutical compositions of near IR, closed chain, sulfo-cyanine dyesand methods for the visualization of tissue under illumination withnear-infrared radiation.

II. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts.

The term “metal ion” as used herein refers to elements of the periodictable that are metallic and that are positively charged as a result ofhaving fewer electrons in the valence shell than is present for theneutral metallic element. Metals that are useful in the presentinvention include metals capable of forming pharmaceutically acceptablecompositions. Useful metals include Li, Na, K, Rb, Cs, Be, Mg, Ca, Sr,and Ba. One of skill in the art will appreciate that the metalsdescribed above can each adopt several different oxidation states. Insome instances, the most stable oxidation state is formed, but otheroxidation states are useful in the present invention.

As used herein, the term “sterile” refers to a system or components of asystem free of infectious agents including bacteria, viruses, andbioactive DNA.

As used herein, the term “non-toxic” refers to the non-occurrence ofdetrimental effects when administered to a vertebrate as a result ofusing a pharmaceutical composition at levels effective for visualizationof tissue under illumination with near-infrared radiation (therapeuticlevels).

The term “unit dosage form” as used herein encompasses any measuredamount that can suitably be used for administering a pharmaceuticalcomposition to a patient. Preferably, the unit dosage form delivers tothe patient an amount between 0.05 and 8 mg/kg, or 0.05 and 5 mg/kg, or0.05 and 1 mg/kg. Suitable dosage ranges delivered to the patient alsoinclude 0.05, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60,0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8 mg/kg. As recognized bythose skilled in the art, when another form (e.g., another salt thepharmaceutical composition) is used in the formulation, the weight canbe adjusted to provide an equivalent amount of the pharmaceuticalcomposition.

As used herein, the term “visualization” refers to methods of obtaininggraphic images of tissue by any means, including illumination withnear-infrared radiation.

As used herein, the term “patient” denotes a mammal, such as a rodent, afeline, a canine, and a primate; most preferably said patient is ahuman.

The term “tissue” as used herein includes, but is not limited to,allogenic or xenogenic bone, neural tissue, fibrous connective tissueincluding tendons and ligaments, cartilage, dura, fascia, pericardia,muscle, heart valves, veins and arteries and other vessels, dermis,adipose tissue, or glandular tissue.

The term “oral dosage form” as used herein refers to its normal meaningin the art (i.e., a pharmaceutical composition in the form of a tablet,capsule, caplet, gelcap, geltab, pill and the like).

The term “injectable dosage form” as used herein refers to its normalmeaning in the art (i.e., refer to a pharmaceutical composition in theform of solutions, suspensions, and emulsions, for example, water orwater/propylene glycol solutions.)

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a patient and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors and colors, and the like. One of skill in the art will recognizethat other pharmaceutical excipients are useful in the presentinvention. Pharmaceutically acceptable carriers include but not limitedto any adjuvants, excipients, glidants, sweeteners, diluents,preservatives, dyes/colorants, flavoring agents, surfactants, wettingagents, dispersing agents, suspending agents, stabilizing agents,isotonic agents, solvents or emulsors.

III. Pharmaceutical Compositions

A. Compounds

The compounds of the present invention comprise dyes of formulas I andII which absorb light at wavelengths in the near-infrared regions of theelectromagnetic spectrum. References herein to the optionally pluralterm “wavelength(s)” indicates that the radiation may be a singlewavelength or a spectrum of radiation having differing wavelengths.

In some embodiments, the present invention provides a sterilepharmaceutical composition comprising a unit dosage form of a compoundhaving the formula:

wherein,

R¹, R², R³, and R⁴ are each independently hydrogen, C₁₋₃ alkyl, or—CO₂T;

X is a single bond, —O—, or —S—;

subscripts a, b, c, and d are each independently an integer from 1 to 6,and

T is a metal ion, H, or a negative charge;

-   wherein, the composition is sterile, non-toxic, and adapted for    administration to a patient for visualization of tissue under    illumination with near-infrared radiation; and    wherein, the unit dosage form of the compound delivers to the    patient an amount between 0.05 and 8 mg/kg.

In some other embodiments, the composition has the formula:

wherein,

R¹, R², R³, and R⁴ are each independently hydrogen, —CH₃, or —CO₂T.

In some other embodiments, the composition has the formula:

In some cases, the composition is adapted for administration to a humanpatient to obtain visualization of human tissue under illumination withnear-infrared radiation wherein the unit dosage form delivers to thepatient an amount between 0.05 and 8 mg/kg. In some cases, thecomposition is dry and a single dose form.

In some cases the composition is lyophilized in a sterile container. Insome cases, the composition is contained within a sterile container,where the container has a machine detectable identifier which isreadable by a medical device.

In some cases, the composition further comprises combination withpharmaceutically acceptable excipients in an oral dosage form. In somecases, the composition further comprises combination withpharmaceutically acceptable carriers in an injectable dosage form. Insome cases, the composition further comprises combination withpharmaceutically acceptable excipients in a dosage form for directapplication to a surgical site.

The compounds of the present invention may exist as salts. The presentinvention includes such salts. Examples of applicable salt forms includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (eg (+)-tartrates,(−)-tartrates or mixtures thereof including racemic mixtures,succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in art.Also included are base addition salts such as sodium, potassium,calcium, ammonium, organic amino, or magnesium salt, or a similar salt.When compounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredacid, either neat or in a suitable inert solvent. Examples of acceptableacid addition salts include those derived from inorganic acids likehydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic,phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from organic acids like acetic, propionic,isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric,lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like. Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Other salts include acid or base salts of the compounds used in themethods of the present invention. Illustrative examples ofpharmaceutically acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid and the like)salts, and quaternary ammonium (methyl iodide, ethyl iodide, and thelike) salts. It is understood that the pharmaceutically acceptable saltsare non-toxic. Additional information on suitable pharmaceuticallyacceptable salts can be found in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., 1985, which isincorporated herein by reference.

Pharmaceutically acceptable salts include salts of the active compoundswhich are prepared with relatively non-toxic acids or bases, dependingon the particular substituents found on the compounds described herein.When compounds of the present invention contain relatively acidicfunctionalities, base addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredbase, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable base addition salts include sodium,potassium, calcium, ammonium, organic amino, or magnesium salt, or asimilar salt. When compounds of the present invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively non-toxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). Certain specific compounds of the present invention containboth basic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

In some embodiments, the present invention provides a pharmaceuticalcomposition including a pharmaceutically acceptable excipient.

B. Synthesis

Exemplary syntheses of the compounds in the pharmaceutical compositionsof the present invention are described in U.S. Pat. Nos. 6,887,854 and6,159,657 and are incorporated herein in their entirety. For example,the synthesis of the compounds in the pharmaceutical compositions of thepresent invention can proceed as follows:

Additionally, some compounds in the pharmaceutical compositions of thepresent invention are commercially available, including DyLight 800(ThermoFisher).

C. Formulation

The compositions of the present invention can be prepared in a widevariety of oral, parenteral and topical dosage forms. Oral preparationsinclude tablets, pills, powder, dragees, capsules, liquids, lozenges,cachets, gels, syrups, slurries, suspensions, etc., suitable foringestion by the patient. The compositions of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compositions described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompositions of the present invention can be administered transdermally.The compositions of this invention can also be administered byintraocular, insufflation, powders, and aerosol formulations (forexamples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol.35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111,1995). Accordingly, the present invention also provides pharmaceuticalcompositions including a pharmaceutically acceptable carrier orexcipient.

For preparing pharmaceutical compositions from the present invention,pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier can beone or more substances, which may also act as diluents, flavoringagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired. The powders and tablets preferably contain from 5% or 10% to70% of the compounds of the present invention.

Suitable solid excipients include, but are not limited to, magnesiumcarbonate; magnesium stearate; talc; pectin; dextrin; starch;tragacanth; a low melting wax; cocoa butter; carbohydrates; sugarsincluding, but not limited to, lactose, sucrose, mannitol, or sorbitol,starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; and gums including arabic and tragacanth; aswell as proteins including, but not limited to, gelatin and collagen. Ifdesired, disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical compositions of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain thecompositions of the present invention mixed with a filler or binderssuch as lactose or starches, lubricants such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, thecompositions of the present invention may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycol with or without stabilizers.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe compositions of the present invention in water and adding suitablecolorants, flavors, stabilizers, and thickening agents as desired.Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing orwetting agents such as a naturally occurring phosphatide (e.g.,lecithin), a condensation product of an alkylene oxide with a fatty acid(e.g., polyoxyethylene stearate), a condensation product of ethyleneoxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partialester derived from a fatty acid and a hexitol (e.g., polyoxyethylenesorbitol mono-oleate), or a condensation product of ethylene oxide witha partial ester derived from fatty acid and a hexitol anhydride (e.g.,polyoxyethylene sorbitan mono-oleate). The aqueous suspension can alsocontain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose, aspartame orsaccharin. Formulations can be adjusted for osmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending the compositions of thepresent invention in a vegetable oil, such as arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin;or a mixture of these. The oil suspensions can contain a thickeningagent, such as beeswax, hard paraffin or cetyl alcohol. Sweeteningagents can be added to provide a palatable oral preparation, such asglycerol, sorbitol or sucrose. These formulations can be preserved bythe addition of an antioxidant such as ascorbic acid. As an example ofan injectable oil vehicle, see Minto, J. Pharmacol. Exp. Ther.281:93-102, 1997. The pharmaceutical formulations of the invention canalso be in the form of oil-in-water emulsions. The oily phase can be avegetable oil or a mineral oil, described above, or a mixture of these.Suitable emulsifying agents include naturally-occurring gums, such asgum acacia and gum tragacanth, naturally occurring phosphatides, such assoybean lecithin, esters or partial esters derived from fatty acids andhexitol anhydrides, such as sorbitan mono-oleate, and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

The compositions of the present invention can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe formulated for administration via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermalroutes afford constant delivery for weeks or months.

In another embodiment, the compositions of the present invention can beformulated for parenteral administration, such as intravenous (IV)administration or administration into a body cavity or lumen of anorgan. The formulations for administration will commonly comprise asolution of the compositions of the present invention dissolved in apharmaceutically acceptable carrier. Among the acceptable vehicles andsolvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid can likewisebe used in the preparation of injectables. These solutions are sterileand generally free of undesirable matter. These formulations may besterilized by conventional, well known techniques including radiation,chemical, heat/pressure, and filtration sterilization techniques. Theformulations may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents, e.g.,sodium acetate, sodium chloride, potassium chloride, calcium chloride,sodium lactate and the like. The concentration of the compositions ofthe present invention in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight, andthe like, in accordance with the particular mode of administrationselected and the patient's needs. For IV administration, the formulationcan be a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents. The sterile injectable preparation canalso be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol.

In another embodiment, the formulations of the compositions of thepresent invention can be delivered by the use of liposomes which fusewith the cellular membrane or are endocytosed, i.e., by employingligands attached to the liposome, or attached directly to theoligonucleotide, that bind to surface membrane protein receptors of thecell resulting in endocytosis. By using liposomes, particularly wherethe liposome surface carries ligands specific for target cells, or areotherwise preferentially directed to a specific organ, one can focus thedelivery of the compositions of the present invention into the targetcells in vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306,1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J.Hosp. Pharm. 46:1576-1587, 1989).

Lipid-based drug delivery systems include lipid solutions, lipidemulsions, lipid dispersions, self-emulsifying drug delivery systems(SEDDS) and self-microemulsifying drug delivery systems (SMEDDS). Inparticular, SEDDS and SMEDDS are isotropic mixtures of lipids,surfactants and co-surfactants that can disperse spontaneously inaqueous media and form fine emulsions (SEDDS) or microemulsions(SMEDDS). Lipids useful in the formulations of the present inventioninclude any natural or synthetic lipids including, but not limited to,sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters,glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®,Capryol®, Capmul®, Captex®, and Peceol®.

The compositions of the present invention are sterile and generally freeof undesirable matter. The compounds and compositions may be sterilizedby conventional, well known techniques including heat/pressure, gasplasma, steam, radiation, chemical, and filtration sterilizationtechniques.

For example, terminal heat sterilization can be used to destroy allviable microorganisms within the final formulation. An autoclave iscommonly used to accomplish terminal heat-sterilization of drug productsin their final packaging. Typical autoclave cycles in the pharmaceuticalindustry to achieve terminal sterilization of the final product are 121°C. for 15 minutes. The compositions of the present invention can beautoclaved at a temperature ranging from 115 to 130° C. for a period oftime ranging from 5 to 40 minutes with acceptable stability. Autoclavingis preferably carried out in the temperature range of 119 to 122° C. fora period of time ranging from 10 to 36 minutes.

The compositions can also be sterilized by dry heat as described byKarlsson, et al., in U.S. Pat. No. 6,392,036, which discloses a methodfor the dry heat sterilization that can be used for drug formulations.The compositions can also be sterilized as described in WO 02/41925 toBreath Limited, which discloses a rapid method, similar topasteurization, for the sterilization of compositions. This methodentails pumping the composition to be sterilized through stainless steelpipes and rapidly raising the temperature of the composition to about130-145° C. for about 2-20 seconds, subsequently followed by rapidcooling in seconds to ambient conditions.

The compositions can also be sterilized by irradiation as described byIllum and Moeller in Arch. Pharm. Chemi. Sci., Ed. 2, 1974, pp.167-174). The compositions can also be sterilized by UV, x-rays, gammarays, e beam radiation, flaming, baking, and chemical sterilization.

Alternatively, sterile pharmaceutical compositions according to thepresent invention may be prepared using aseptic processing techniques.Aseptic filling is ordinarily used to prepare drug products that willnot withstand heat sterilization, but in which all of the ingredientsare sterile. Sterility is maintained by using sterile materials and acontrolled working environment. All containers and apparatus aresterilized, preferably by heat sterilization, prior to filling. Thecontainer (e.g., vial, ampoule, infusion bag, bottle, or syringe) arethen filled under aseptic conditions.

The compounds and compositions of the present invention are non-toxicand generally free of detrimental effects when administered to avertebrate at levels effective for visualization of tissue underillumination with near-infrared radiation. Toxicity of the compounds andcompositions of the present invention can be assessed by measuring theireffects on a target (organism, organ, tissue or cell). Becauseindividual targets typically have different levels of response to thesame dose of a compound, a population-level measure of toxicity is oftenused which relates the probabilities of an outcome for a givenindividual in a population. Toxicology of compounds can be determined byconventional, well known techniques including in vitro (outside of aliving organism) and in vivo (inside of a living organism) studies.

For example, determination of metabolic stability is commonly examinedwhen assessing the toxicity of a compound as it is one of several majordeterminates in defining the oral bioavailability and systemic clearanceof a compound. After a compound is administered orally, it firstencounters metabolic enzymes in the gastrointestinal lumen as well as inthe intestinal epithelium. After it is absorbed into the bloodstreamthrough the intestinal epithelium, it is first delivered to the livervia the portal vein. A compound can be effectively cleared by intestinalor hepatic metabolism before it reaches systemic circulation, a processknown as first pass metabolism. The stability of a compound towardsmetabolism within the liver as well as extrahepatic tissues willultimately determine the concentration of the compound found in thesystemic circulation and affect its half-life and residence time withinthe body. Cytochrome P450 (CYP) enzymes are found primarily in the liverbut also in the intestinal wall, lungs, kidneys and other extrahepaticorgans and are the major enzymes involved in compound metabolism. Manycompounds undergo deactivation by CYPs, either directly or byfacilitated excretion from the body. Also, many compounds arebioactivated by CYPs to form their active compounds. Thus, determiningthe reactivity of a compound to CYP enzymes is commonly used to assessmetabolic stability of a compound.

The Ames reverse mutation Assay is another common toxicology assay forassessing the toxicity of a compound. The Ames Assay, utilizes severaldifferent tester strains, each with a distinct mutation in one of thegenes comprising the histidine (his) biosynthetic operon (Ames, B. N.,et al., (1975) Mutation Res. 31:347-363). The detection of revertant(i.e., mutant) bacteria in test samples that are capable of growth inthe absence of histidine indicates that the compound under evaluation ischaracterized by genotoxic (i.e. mutagenic) activity. The Ames Assay iscapable of detecting several different types of mutations (geneticdamage) which may occur in one or more of the tester strains. Thepractice of using an in vitro bacterial assay to evaluate the genotoxicactivity of drug candidates is based on the prediction that a substancethat is mutagenic in a bacterium is likely to be carcinogenic inlaboratory animals, and by extension may be carcinogenic or mutagenic tohumans.

In addition, the human ether-a-go-go related gene (hERG) assay can beused to evaluate the potential cardiotoxicity of a compound.Cardiotoxicity can arise when the QT interval is prolonged leading to anelevated risk of life-threatening arrhythmias. The QT interval is ameasure of the time between the start of the Q wave and the end of the Twave in the heart's electrical cycle. The QT interval representselectrical depolarization and repolarization of the ventricles. Alengthened QT interval has most commonly been associated with loss ofcurrent through hERG potassium ion channels due to direct block of theion channel by drugs or by inhibition of the plasma membrane expressionof the channel protein (Su et al. J. Biomol Screen 2011, 16, 101-111).Thus, an in vitro hERG screening assay can be used to detect disruptionor inhibition of the hERG membrane trafficking function and assesspotential cardiotoxicity of a compound.

Other methods of assessing the toxicity of compounds include in vivostudies which administer relatively large doses of a test compound to agroup of animals to determine the level which is lethal to a percentageof the population (mean lethal dose LD₅₀ or mean lethal concentrationLC₅₀). Toxicity of a compound can also be assessed in vivo by examiningwhether a compound produces statistically significant negative effectson cardiac, blood pressure, central nervous system (CNS), body weight,food intake, gross or microscopic pathology, clinical pathology, orrespiratory measures in an animal.

For example, in a set of in vitro studies evaluating the metabolicstability of a composition of formula III, it was shown that thecompound appears to be stable in rat, dog and human plasma, does notappear to be broken down into metabolites, and does not show anysignificant reactivity to 9 major CYP liver enzymes. Additionally, thecomposition of formula III does not show any mutagenicity at any of thetested doses in the Ames reverse mutation assay, a widely used in vitromethod that determines the ability of a chemical to cause mutations inDNA. Further safety pharmacology was assessed in vitro using the humanether-a-go-go related gene (hERG) assay for determining possiblecardio-toxic effects. Studies utilizing this assay determined that thecomposition of formula III shows only a small (11%) inhibition of hERGfunction at the highest tested concentration (30 times higher thanlevels effective for visualization), indicating even at thisconcentration the composition of formula III is unlikely to translateinto any clinically threatening physiological cardiac changes.

Furthermore, toxicology of the composition of formula III was alsoinvestigated in non-clinical in vivo studies in both rats and dogs. Astudy evaluating the effects of the composition of formula III oncardiac and respiratory safety in radio-telemetry monitored dogs showedno statistically significant negative effects on cardiac, blood pressureor respiratory measures at doses up to 80 times higher than thoserequired for visualization. Additional safety pharmacology performedover 28 days in rats indicated doses as high as 160 times visualizationlevels per day showed no biologically meaningful effects on the centralnervous system (CNS). Finally, a series of toxicology studies in dogsshowed doses as high as 100 times visualization levels showed nosignificant effects on body weight, food intake, gross or microscopicpathology as well as clinical pathology (clinical serum chemistry,hematology, coagulation and urinalysis). No mortality was observed atany tested dose of the composition of formula III in rat, dog or piganimal models.

In some embodiments, the compositions of the present invention can belyophilized in a sterile container for convenient dry storage andtransport. A ready-to-use preparation can subsequently be made byreconstituting the lyophilized compositions with sterile water. Theterms “lyophilization,” “lyophilized,” and “freeze-dried” refer to aprocess by which the material to be dried is first frozen and then theice or frozen solvent is removed by sublimation in a vacuum environment.An excipient may be included in pre-lyophilized formulations to enhancestability of the lyophilized product upon storage.

In some embodiments, the composition can be contained within a sterilecontainer, where the container has a machine detectable identifier whichis readable by a medical device. As used herein, “machine detectableidentifier” includes identifiers visible or detectable by machinesincluding medical devices. In some instances, the medical device is atele-surgical system. Machine detectable identifiers may facilitate theaccess or utilization of information that is directly encoded in themachine detectable identifier, or stored elsewhere. Examples of machinedetectable identifiers include microchips, radio frequencyidentification (RFID) tags, barcodes (e.g., 1-dimensional or2-dimensional barcode), data matrices, quick-response (QR) codes, andholograms. One of skill in the art will recognize that other machinedetectable identifiers are useful in the present invention.

In some cases, the machine detectable identifier can include amicrochip, an integrated circuit (IC) chip, or an electronic signal froma microchip which is detectable and/or readable by a computer systemwhich is in communication with the medical device. In some cases, themachine detectable identifier includes a radio frequency identification(RFID) tag. RFID tags are sometimes called as transponders. RFID tagsgenerally are devices formed of an IC chip, an antenna, an adhesivematerial, and are used for transmitting or receiving predetermined datawith an external reader or interrogator. RFID tags can transmit orreceive data with a reader by using a contactless method. According tothe amplitude of a used frequency, inductive coupling, backscattering,and surface acoustic wave (SAW) may be used. Using electromagneticwaves, data may be transmitted or received to or from a reader by usinga full duplex method, a half duplex (HDX) method, or a sequential (SEQ)method.

In some cases, the machine detectable identifier can include a barcode.Barcodes include any machine-readable format, including one-dimensionaland two-dimensional formats. One-dimensional formats include, forexample, Universal Product Code (UPC) and Reduced Space Symbology (RSS).Two-dimensional formats, or machine-readable matrices, include forexample, Quick Response (QR) Code and Data Matrix.

In some cases, the medical device can be configured to detect themachine detectable identifier. In one example, the medical device is atele-surgical system that includes a special imaging mode (e.g., afluorescence imaging mode) for use with dyes such as those described inthis disclosure. One example of a tele-surgical system that includes afluorescence imaging mode is described in U.S. Pat. No. 8,169,468,entitled “Augmented Stereoscopic Visualization for a Surgical Robot,”which is hereby incorporated in its entirety herein. In some cases,medical devices can incorporate an imaging device that can scan, read,view, or otherwise detect a machine detectable identifier that isdisplayed to the imaging device. In one aspect, the medical device willpermit a user to access the fluorescence imaging mode of the medicaldevice only if the medical device detects the presence of a knownmachine detectable identifier that corresponds to a dye identified asbeing compatible for use with the medical device. In contrast, if themedical device does not detect a known machine detectable identifier,the medical device will not permit a user to access the fluorescenceimaging mode and associated functionality. Imaging devices can includeoptical scanners, barcode readers, cameras, and imaging devicescontained within a tele-surgical system such as an endoscope.Information associated with the machine detectable identifier can thenbe retrieved by the medical device using an imaging device. Upondetection of the identifier, an automatic process may be launched tocause a predetermined action to occur, or certain data to be retrievedor accessed. The information encoded onto the machine detectableidentifier may include instructions for triggering an action, such asadministering a composition of the present invention to a patient. Insome embodiments, the machine detectable identifier includes unencryptede-pedigree information in the desired format. The e-pedigree informationcan include, for example, lot, potency, expiration, national drug code,electronic product code, manufacturer, distributor, wholesaler, pharmacyand/or a unique identifier of the salable unit.

In some embodiments, the sterile container having a machine detectableidentifier includes a fluid outlet configured to mate with the medicaldevice. In some cases, the fluid outlet of the machine detectableidentifier is mechanically affixed to the medical device.

C. Administration

The compounds and compositions of the present invention can be deliveredby any suitable means, including oral, parenteral and topical methods.Transdermal administration methods, by a topical route, can beformulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the compounds and compositions of the presentinvention. The unit dosage form can be a packaged preparation, thepackage containing discrete quantities of preparation, such as packetedtablets, capsules, and powders in vials or ampoules. Also, the unitdosage form can be a capsule, tablet, cachet, or lozenge itself, or itcan be the appropriate number of any of these in packaged form.

In some embodiments, co-administration can be accomplished byco-formulation, i.e., preparing a single pharmaceutical compositionincluding the compounds and compositions of the present invention andany other agent. Alternatively, the various components can be formulatedseparately.

The compositions of the present invention, and any other agents, can bepresent in any suitable amount, and can depend on various factorsincluding, but not limited to, weight and age of the patient, state ofthe disease, etc. Suitable dosage ranges deliver to the patient anamount from about 0.05 and 8 mg/kg, or about 0.05 and 5 mg/kg, or about0.05 and 1 mg/kg. Suitable dosage ranges also deliver to the patient theamount of 0.05, 0.10, 0.20, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60,0.65, 0.70, 0.75, 0.80, 0.90, 1, 2, 4, 6, or 8 mg/kg.

The composition can also contain other compatible compositions. Thecompositions described herein can be used in combination with oneanother, with other active compositions known to be useful forvisualization of tissue under illumination with near-infrared radiation,or with compositions that may not be effective alone, but may contributeto the efficacy of the active composition.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding equivalents of thefeatures shown and described, or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention claimed. Moreover, any one or more features of any embodimentof the invention may be combined with any one or more other features ofany other embodiment of the invention, without departing from the scopeof the invention. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

IV. Methods for Visualization of Tissue

The present invention generally provides novel, sterile, and non-toxicpharmaceutical compositions of near IR, closed chain, sulfo-cyanine dyesand methods for the visualization of tissue under illumination withnear-infrared radiation.

In a first embodiment, the present invention provides a sterilepharmaceutical composition comprising a unit dosage form of a compoundhaving the formula:

wherein,

R¹, R², R³, and R⁴ are each independently hydrogen, C₁₋₃ alkyl, or—CO₂T;

X is a single bond, —O—, or —S—;

subscripts a, b, c, and d are each independently an integer from 1 to 6,and

T is a metal ion, H, or a negative charge;

-   wherein, the composition is sterile, non-toxic, and adapted for    administration to a patient for visualization of tissue under    illumination with near-infrared radiation; and    wherein, the unit dosage form of the compound delivers to the    patient an amount between 0.05 and 8 mg/kg.

In some embodiments, the present invention provides a use of thecomposition adapted for administration to a patient to obtainvisualization of tissue under illumination with near-infrared radiationwherein the unit dosage form delivers to the patient an amount between0.05 and 8 mg/kg. In some cases, the use is adapted for administrationto a human patient to obtain visualization of human tissue underillumination with near-infrared radiation wherein the unit dosage formdelivers to the patient an amount between 0.05 and 8 mg/kg.

In some embodiments, the present invention provides a method forvisualization of tissue, the method comprising administering thecomposition to a patient, imaging the tissue under illumination withnear-infrared radiation, and obtaining at least one image of tissue fromthe patient using the composition. In some cases, the method furthercomprises obtaining the image during administration, afteradministration, or both during and after administration of thecomposition.

In some cases, the method further comprises intravenously injecting thecomposition into a patient. In some cases, the composition is injectedinto a circulatory system.

In some cases, the method further comprises visualizing a patient areaon which surgery is or will be performed, or for viewing a patient areaotherwise being examined by a medical professional. In some cases, themethod further comprises performing a surgical procedure on the patientareas based on the visualization of the surgical area. In some cases,the method further comprises viewing a patient area on which anophthalmic, arthroscopic, laparoscopic, cardiothoracic, muscular, orneuro procedure is or will be performed. In some cases, the methodfurther comprises obtaining ex vivo images of at least a portion of thepatient.

In some cases, the method further comprises the use of compositions ofthe present invention in combination with the da Vinci Surgical System'sFirefly fluorescence, in providing an augmented view that enhances thedifficult-to-visualize tissue to reduce the injury rate and possiblyspeed up surgery by providing the surgeon with confidence and securitythat he/she is not causing injury. Surgeons often have a difficult timelocating the tubular structure known as the ureter, which carries urinefrom the kidneys to the bladder, during minimally invasive gynecologicprocedures such as benign and malignant hysterectomy due to the methodof approaching and visualizing the relevant anatomy. The incidence rateof ureteral injury is 1-3%, amounting to 5000-15000 women annually inthe US. The injury rate is even higher in colorectal surgeries, on theorder of 6-8% and the ureter is often cut if the surgeon does not takeextreme care in doing a meticulous dissection. In cases with significantadhesions or fibrosis from prior surgeries, radiation therapy or otherinterventions, the task of identification can be much more difficult andtime consuming. In complex urological cases such as pyeloplasty torelieve uretero-pelvic junction obstructions, visualization of theureter can be difficult and an imaging agent such as compositions of thepresent invention can help the surgeon identify normal or aberrantanatomy.

V. Examples Example 1. Visualization of Kidney and Ureter UnderIllumination

20 mg of a compound of formula III in 10 ml of sterile water wasadministered intravenously to five pigs. Laparoscopic ports were placedand the da Vinci Surgical System was connected to the ports. Theendoscope of the system was directed at the kidney of the pig, and laserexcitation at approximately 800 nm was used to excite the composition offormula III within the ureter (FIG. 1). A small amount of blue and greenlight were also emitted in order to allow visualization of thebackground anatomy. Approximately 10 minutes after administration, thecomposition of formula III was seen exiting the kidney via the ureter,on its way to the bladder for excretion (FIGS. 2A and 2B).

Example 2. Visualization of Kidney and Ureter Under Illumination

An augmented view to enhance difficult-to-visualize kidneys ureters in apatient can be achieved by intravenously administering 20 mg of acompound of formula III in 10 ml of sterile water to a 60 kg humanpatient 15 minutes prior to desired visualization. The endoscope of thesystem can be directed at the kidney of the patient, and laserexcitation at approximately 800 nm can be used to excite the compositionof formula III within the ureter. A small amount of blue and green lightcan also be emitted in order to allow visualization of the backgroundanatomy. Approximately 15 minutes after administration, visualization ofthe ureter can be achieved as the composition of formula III exits thekidney via the ureter, on its way to the bladder for excretion.

What is claimed is:
 1. A method of visualizing tissue in a patient, themethod comprising the step of administering a sterile pharmaceuticalcomposition comprising a unit dosage form of a compound having theformula:

wherein, R¹, R², R³, and R⁴ are each independently hydrogen, C₁₋₃ alkyl,or —CO₂T; X is a single bond, —O—, or —S—; subscripts a, b, c, and d areeach independently an integer from 1 to 6, and T is a metal ion, H, or anegative charge; wherein the composition is non-toxic, and wherein theunit dosage form delivers to the patient an amount sufficient tovisualize tissue; and further comprising imaging the tissue underillumination with near-infrared radiation, and obtaining at least oneimage of the tissue from the patient using the composition.
 2. Themethod of claim 1 wherein the unit dosage form delivers to the patientan amount between 0.05 and 8 mg/kg.
 3. The method of claim 1 furthercomprising obtaining the image during administration, afteradministration, or both during and after administration of thecomposition.
 4. The method of claim 1 where the step of administering asterile pharmaceutical composition is by intravenous injection.
 5. Themethod of claim 1 where the composition is injected into a circulatorysystem.
 6. The method of claim 1 further comprising visualizing apatient area on which surgery is or will be performed, or for viewing apatient area otherwise being examined by a medical professional.
 7. Themethod of claim 6 further comprising performing a surgical procedure onthe patient area based on the visualization of the surgical area.
 8. Themethod of claim 6 further comprising viewing a patient area on which anophthalmic, arthroscopic, laparoscopic, cardiothoracic, muscular, orneuro procedure is or will be performed.
 9. The method of claim 1further comprising obtaining ex vivo images of at least a portion of thepatient.
 10. The method of claim 1 wherein the compound has the formula:

wherein, R¹, R², R³, and R⁴ are each independently hydrogen, —CH₃, or—CO₂T.
 11. The method of claim 1 wherein the compound is: